Colloids and Surfaces A: Physicochem. Eng.

Aspects 372 (2010) 4854

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Colloids and Surfaces A: Physicochemical and Engineering Aspects

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Multiple W/O/W emulsionsUsing the required HLB for emulsier evaluation

T. Schmidts, D. Dobler, A.-C. Guldan, N. Paulus, F. Runkel
Institute of Biopharmaceutical Technology (IBPT), University of Applied Sciences Giessen-Friedberg, Wiesenstr. 14, 35390 Giessen, Germany

a r t i c l e

i n f o

a b s t r a c t
Stable emulsions are best formulated with emulsiers or combinations of emulsiers, which possess HLB values close to the required HLB of the oil phase. In this work, we have investigated the application of this established method to the development of multiple emulsions. This is of particular interest, since multiple emulsions are highly sensitive in terms of variations of the individual components as a result of the presence of two thermodynamically unstable interfaces. However, multiple W/O/W emulsions are potential skin delivery systems for water-soluble active pharmaceutical ingredients as a result of their pronounced encapsulation properties. Firstly, a suitable primary emulsion was developed based on required HLB determinations of the investigated oils. Secondly and based on the required HLB, multiple W/O/W emulsions were developed using the most appropriate primary emulsion and 1% of hydrophilic emulsier blends in order to stabilise the second interface. In order to nd the appropriate mixtures of hydrophilic emulsiers, the required HLB for the primary W/O emulsion was determined using two different chemical classes of emulsier blends, i.e. polyethoxylated ethers and polyethoxylated esters. The physicochemical parameters of the formulations were characterised by means of rheological measurements, droplet size and creaming volume observations as well as by means of conductivity analysis. As discovered, all methods are appropriate for determining the required HLB determination with the exception of the rheological data. Referring to the primary emulsions tested, required HLB values of 4.34.7 using parafn as the oil phase resulted in stable emulsions. Irrespective of the emulsiers used, the nest droplets, lowest conductivity and minimal creaming volume were obtained for the multiple emulsions at required HLB values between 15 and 15.5 using parafn as the oil phase. What is more, using a polyethoxylated ether instead of a polyethoxylated fatty acid ester resulted in more stable multiple emulsions. 2010 Elsevier B.V. All rights reserved.

Article history: Received 27 July 2010 Received in revised form 17 September 2010 Accepted 20 September 2010 Available online 25 September 2010 Keywords: W/O/W multiple emulsions HLB Hydrophilic surfactant Required HLB Electrolyte encapsulation

1. Introduction Owing to their distinct structure and properties, multiple emulsions are of particular interest for several drug delivery approaches, including carrier systems for the dermal application of pharmaceutical drugs [15]. Carrier systems in particular are developed by empirical means. These empirical procedures are often extremely time-consuming, since particularly sensitive carrier systems require a well-dened ratio of ingredients [6]. It is therefore of great importance to incorporate the physicochemical properties of the selected constituents at an early stage of the formulation development process. Two of these major parameters are the hydrophiliclipophilic balance (HLB) value of the emulsiers used and, determined by empirical tests, the required HLB (rHLB) value of the oil phase investigated. Using these parameters, the number of experiments can be reduced early during the formulation screening stage [710].

Corresponding author. Tel.: +49 641 3092550. E-mail address: Frank.Runkel@tg.fh-giessen.de (F. Runkel). 0927-7757/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.09.025

Grifn rst established the hydrophiliclipophilic balance (HLB) system to classify non-ionic surfactants [7]. According to Grifn, low HLB values are ascribed to lipophilic surfactants, whereas hydrophilic surfactants are considered to possess high HLB values. With regard to this system, W/O emulsions are obtained using surfactants with HLB values ranging between 3 and 8 and O/W emulsions are formed using surfactants with HLB values between 9 and 12. Stable emulsions are best formulated with emulsiers or combinations of emulsiers, which possess HLB values close to the so-called rHLB of the oil or oil phase used. In order to determine the rHLB of an oil or oil phase, emulsions are produced with different ratios of emulsier blends, representing various HLBs, and are investigated in respect of their separating properties [24]. However, not only the HLB value but also the chemical type of emulsier can inuence the stability of emulsions. For example, depending on the emulsier systems used and the phase obtained, the required HLB for parafn oil is proposed by some authors to range between 10 [7] and 12 [9] for simple O/W emulsions. For simple W/O emulsions, it is thought to be 4 [7]. Water-in-oil-in-water (W/O/W) multiple emulsions are systems where both W/O and O/W emulsions appear simultaneously.

T. Schmidts et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 372 (2010) 4854 Table 1 Results of the production of primary W/O emulsions. Numbers in brackets represent the HLB value of the lipophilic surfactant. Sorbitan trioleate (1.8) Medium-chain triglycerides Isopropyl palmitate Octyldodecanol Light parafn Heavy parafn No emulsion No emulsion No emulsion No emulsion No emulsion Sorbitan sesquioleate (3.7) No emulsion Phase separation Phase separation Phase separation Emulsion Sorbitan monooleate (4.3) No emulsion Phase separation Phase separation Emulsion Emulsion Sorbitan monostearate (4.7) No emulsion Phase separation Phase separation Emulsion Emulsion

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Sorbitan laurate (8.7) No emulsion No emulsion No emulsion No emulsion Phase separation

The main problem regarding their stability, however, is the presence of two thermodynamically unstable interfaces, i.e. the W/O interface of the primary emulsion and the O/W interface of the multiple emulsion. Two different emulsiers are therefore necessary for their stabilisation: one with a low HLB for the W/O interface and a second one with a high HLB for the O/W interface. The effect of the nature and quantity of both emulsiers on the properties of multiple emulsions is discussed in several works [6,1114]. When choosing lipophilic surfactants and determining the rHLB for the primary W/O emulsion of multiple emulsions, surfactants for interface stabilisation can be screened in the same way as is done for simple W/O emulsions. However, the determination of a required HLB for W/O/W multiple emulsions and thus the selection of hydrophilic emulsiers, which are necessary to stabilise the second interface and form a multiple emulsion, is more complex. It has been shown that both lipophilic and hydrophilic emulsiers can be absorbed on O/W interfaces [15]. The HLB value of the O/W interface of multiple emulsions is therefore, if both hydrophilic and lipophilic emulsiers are added, the sum of the HLB values of all absorbed surfactants. The amount of absorbed lipophilic emulsier to the O/W interface is however not dened and dependent on its total amount in the formulation and the phase volume ratio, for example. Magdassi et al. [15] discovered that the ideal HLB value for hydrophilic emulsiers increases when the concentration of the lipophilic emulsier added is higher or when the amount of the hydrophilic emulsier is lowered. The purpose of our research was rst to screen for an appropriate primary W/O emulsion using rHLB considerations. Secondly, in order to determine the rHLB, which will permit the most appropriate primary emulsion to be incorporated into the multiple emulsion, W/O/W emulsions were prepared with different blends of hydrophilic emulsiers representing a range of HLB values and the properties and long-time stability of the multiple W/O/W emulsions obtained were investigated. The following parameters were used to determinate the stability of the formulation as well as the ideal rHLB allowing the primary W/O emulsion to be incorporated into the multiple emulsions: conductivity, multiple droplet size, creaming volume and viscosity. All of these methods have been evaluated and compared. Their suitability for determining the rHLB value of multiple W/O/W emulsions was investigated.

PEG-8 stearate was supplied by Croda GmbH (Kaldenkirchen, Germany). A NaCl solution (0.1 M) was used as the inner water phase, NaCl was supplied in Ph. Eur. grade by Merck KG (Darmstadt, Germany). 2.2. Preparation of the emulsions Multiple emulsions were prepared using a 2-step procedure, as reported by Matsumoto et al. [16]. In the rst step, the primary W/O emulsion is prepared and in the second step, 40% of the primary emulsion is dispersed in an aqueous solution of the hydrophilic emulsier in order to obtain a multiple emulsion. The primary W/O emulsion was prepared by adding the aqueous phase (50%) containing 0.1 M NaCl solution to the oil phase (50%). The oil phase consisted of 20% lipophilic emulsier and 80% oil. The compositions of the primary emulsions and the multiple emulsions are shown in Tables 1 and 2, respectively. The two phases were heated separately to approx. 7075 C. After adding the water phase to the oil phase, the emulsion was homogenised for 2 min using a rotor/stator homogeniser Diax 600 (Heidolph, Germany) at 9500 rpm. In the second step, the primary emulsion was cooled down to room temperature and then slowly added to the outer water phase while the system was stirred at 1200 rpm using a EUROSTAR digital stirrer (IKA, Germany) until a homogeneous emulsion was produced. The obtained phase ratio W1 :O:W2 was 1:1:3. 2.3. Conductometric analysis Conductivity measurements were carried out using a WTW Microprocessor Conductivity Meter LF 96 (WTW, Germany) at room temperature. Measurements were performed directly in the undiluted emulsion (mean S.D., n = 3). To estimate the released mass fraction of NaCl, a calibration curve was plotted. 2.4. Microscopic observation The W/O/W multiple emulsions were analysed using an optical immersion microscope TR 300 connected to a DV 2B camera (VWR, Germany) at 1000 magnifying power (oil immersion). This method was used in order to facilitate a standardised quality control procedure as well verication of the multiple emulsions. 2.5. Droplet size measurement Mean water droplet size (z-average) in the primary W/O emulsion was determined by dynamic light scattering (High Performance Particle Sizer (HPPS), Malvern Instruments, UK). Samples
Table 2 Composition of multiple W/O/W emulsions. 0.1 M NaCl solution Heavy parafn oil Sorbitan monoolete Aqua dest. Hydrophilic emulsier 20% 16% 4% 59% 1% Inner water phase Oil phase

2. Materials and methods 2.1. Materials The following chemicals were used to prepare the emulsions. All chemicals are of Ph. Eur. quality: Heavy and light parafn, the oils isopropyl palmitate, octyldodecanol and medium-chain triglycerides were supplied by Fagron GmbH Co. KG (Barsbttel, Germany). The lipophilic surfactants sorbitan monooleate, sorbitan sesquioleate, sorbitan monostearate, glyceryl stearate and sorbitan trioleate were supplied by Croda GmbH (Kaldenkirchen, Germany). The hydrophilic surfactant polysorbate 20 was supplied by Caelo GmbH (Hilden, Germany), steareth-10 and ceteareth-30 were obtained from Cognis GmbH (Monheim, Germany), whereas

Outer water phase

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were diluted 1:1000 using light parafn oil (viscosity: 33 mPa s) prior to measurement. Oil droplet size and distribution in multiple W/O/W emulsions were determined using a laser diffraction particle size analyser (Mastersizer S, Malvern Instruments, England). The fundamental size distribution obtained with this technique is based on volume distribution. The particle size distribution was calculated according to the Mie theory. Measurements were performed directly after dilution in iso-osmotic glucose solution (0.1 M), thereby taking into account the osmotic pressure of the internal aqueous phase. (mean S.D., n = 3). Microscopic observations revealed that the bimodal particle size distribution obtained by static light scattering measurements can be attributed to the occurrence of simple oil droplets without an encapsulated water phase and oil droplets with multiplicity (containing inner water droplets). The peak size of the larger multiple droplets was therefore chosen for characterising the W/O/W multiple emulsions. 2.6. Rheological measurement Rheological analysis was performed at 25 C using a RheoStress 300 Rheometer (Thermo Haake, France) with a 2 cm diameter cone and plate geometry measuring system. The geometry had a 2 angle. The apparent viscosity was measured over a shear rate of 0.1100 s1 . The results are presented as mean values (mean S.D., n = 3). 2.7. Degree of creaming A 40 ml sample of emulsion was poured into a 50 ml graduated cylinder immediately prior to preparation. The volume ratio of the separated aqueous phase to the total volume of the emulsion was determined at room temperature (22 C) as a function of time over 12 weeks. The values obtained were averages of three determinations. The results are presented as mean values (mean S.D., n = 3). 3. Results and discussion 3.1. Development of W/O primary emulsion A basic principle of stable multiple W/O/W emulsion formation and efcient drug encapsulation is the creation of homogeneous and small inner water droplets (W1 < 1 m) [17]. A uniform distribution of droplet size counteracts Ostwald ripening, a process in which large droplets grow at the expense of small ones [18]. By way of contrast, decreasing droplet size results in an increase in Laplace pressure, which can induce coalescence. Laplace pressure can be compensated for by a small quantity of salt in the W1 droplets [19]. However, the salt concentration in W1 is critical due to the osmotic gradient between the inner (W1 ) and outer (W2 ) water phase. Therefore, the electrolyte concentration should be sufciently high so as to compensate for Laplace pressure but, at the same time, be low enough so as to inhibit osmotic swelling of the W1 droplets in the multiple emulsion [17]. Another crucial parameter for stable W/O/W emulsions is the selection of the appropriate type and amount of surfactants as well as the prevention of negative interactions between the W/O and the O/W emulsier. It was found that increasing the oil-soluble surfactant concentration can hamper the exchange of water molecules [11] as well as the transport of NaCl ions [18] between the two water phases. Moreover, it is preferable to use low molecular emulsiers for W/O (e.g. polyglycerol polyricinoleate or sorbitan monooleate) in combination with a high molecular emulsier (e.g. proteins) [20] or a lipophilic polymeric emulsier for the outer water phase [21]. Furthermore, the instability of W/O/W emulsions can be increased

by the use of unsaturated oils in the oil phase, which prevents a close packed, condensed interfacial lm [22,23]. The present work pursued the approach of the rHLB for the development of appropriate primary W/O emulsions. Therefore, several saturated oils (isopropyl palmitate, octyldodecanol, medium-chain triglycerides, light parafn and heavy parafn) and ve lipophilic surfactants with HLB values ranging from 1.8 to 8.7 were chosen for the preparation of primary W/O emulsions. The surfactants are of the same surfactant class (sorbitan ester) with different side chains resulting in various HLB values. All primary W/O emulsions consisted of 50% of an aqueous phase (0.1 M NaCl solution) and 50% of an oil phase (80% oil and 20% lipophilic surfactant). It was found that, using medium-chain triglycerides, isopropyl palmitate or octyldodecanol, neither homogeneous nor stable (phase separation occurs in a few minutes) formulations were obtained. Both parafn oils, particularly the heavy parafn, formed more stable formulations (Table 1). More precisely, light parafn resulted only with sorbitan monooleate and sorbitan monostearate in formulations that were stable for approximately 35 h. Heavy parafn exhibited the same behaviour for these two surfactants, but additionally resulted in a metastable W/O emulsion (2 h) using sorbitan sesquioleate. Referring to the results presented in Table 1, one can conclude that, using a 0.1 M NaCl solution as the water phase, the rHLB values for light and heavy parafn range between 4.3 and 4.7 and 3.7 and 4.7, respectively. The most stable emulsion was obtained using heavy parafn stabilised with sorbitan monooleate. The viscosity of this primary emulsion was 1.9 0.1 Pa s and the mean water droplet size was approximately 650 nm. In comparison to the other tested oils in this work, parafn oil is strong non-polar. Therefore, the emulsions containing this oil are probably more stable. Furthermore, the rHLB of parafn oil is about 4 [24] and consequently emulsiers with an HLB of approximately 4 will create the most stable formulations. The stability of W/O/W emulsions can be further improved by the addition of a thickener to the inner water phase [25]. Therefore, we investigated the inuence of the two thickeners cetyl palmitate (2%) and hydrogenated castor oil (1%) on the stability of the primary emulsion prepared with heavy parafn and sorbitan monooleate. Both thickeners increased the viscosity of the W/O emulsion (4 Pa s) and its stability. No phase separation occurred over at least seven days. Nevertheless, from our experience using these more highly viscous primary emulsions for the production of W/O/W emulsions resulted in phase inversion into simple W/O emulsions. By way of contrast, less stable and low-viscous primary W/O emulsions (without thickener) produced stable W/O/W emulsions. Therefore, the formulation consisting of heavy parafn sorbitan monooleate and 0.1 M NaCl solution was used as the primary emulsion for the following study. 3.2. Multiple emulsion development using rHLB considerations Multiple emulsions have potential for many applications. Their use as drug delivery systems in particular seems to be most promising. They can be developed as systems for oral, intravenous [26] or topical [4] applications. The advantages of multiple emulsions as opposed to simple W/O or O/W emulsions are based on the following properties: - protection of drugs incorporated in the inner phase; - controlled release of drugs; - ability to integrate incompatible substances into the different phases.

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Despite their advantages, multiple emulsions are, in the main, extremely unstable. Two processes in particular must be borne in mind when it comes to multiple emulsion destabilisation: the coagulation of multiple droplets and multiple drop break-down. Previous studies have shown that the composition of multiple emulsions, especially the selection of appropriate emulsiers, plays a major role in terms of producing stable formulations [6]. Referring to emulsiers, the signicance of an ideal chemical composition and HLB value for producing simple emulsions was studied in the 50s by Grifn [7]. However, in the case of multiple W/O/W emulsions, these results can only be applied directly to primary W/O emulsions. Multiple W/O/W emulsions always contain two different emulsiers (lipophilic and hydrophilic), the distribution of which in the emulsion is, in the main, not precisely known and dependent on the total amount of emulsiers in the formulation and the phase volume ratio, for example. Therefore, the ideal HLB for hydrophilic emulsiers varies from system to system and has to be determined on an individual basis for each formulation. In the present work, the rHLB of hydrophilic emulsiers was determined by the constant composition of W/O/W formulations. To verify the results, two different emulsier combinations were used: steareth-10 (HLB = 12.4) in combination with cetosteareth30 (HLB = 17) and PEG-8 stearate (HLB = 10.8) in combination with polysorbate 20 (HLB = 16.7). The emulsiers represent chemical substances with different functional groups. Steareth-10 and cetosteareth-30 are polyethoxylated fatty alcohol ethers, whereas PEG-8 stearate is a polyethoxylated fatty acid ester. Polysorbate 20 is polyethoxylated sorbitan and a fatty acid ester, where both functional groups, i.e. esters and ethers, are present. The chemical composition of emulsiers has no inuence on the general trends in the HLB range. Therefore, both emulsier mixes were able to be used for determining the rHLB in order to further optimise the formulations. Emulsions with different physical properties (droplet size, viscosity) are nevertheless to be expected. Therefore, the inuence of the chemical composition of the emulsiers on emulsion stability was also able to be determined in the process. The composition of the multiple emulsions is presented in Table 2. Based on the results of the development of the primary W/O emulsion, sorbitan monooleate was chosen as the lipophilic emulsier and heavy parafn as the oil. In the second preparation step, the emulsier mixes mentioned above were used as the hydrophilic emulsier: steareth-10 (HLB = 12.4) in combination with cetosteareth-30 (HLB = 17) and PEG-8 stearate (HLB = 10.8) in combination with polysorbate 20 (HLB = 16.7). The HLBs were adjusted by mixing the emulsiers in order to obtain HLB values ranging between 13 and 17 (0.5 unit step). For the steareth10/cetosteareth-30 emulsier system, the emulsions were prepared 3 times in order to determine the reproducibility of the preparation process. Repetition of the experiments is particularly important, as the properties of W/O/W emulsions are highly sensitive in relation to the parameters applied during their preparation. Referring to the samples studied, relevant differences in the properties of the obtained formulations with the same composition were also found. The differences are probably associated with the manual production process, as the individual steps were not able to be carried out in identical manner. However, a general trend in formulation properties (droplet size, viscosity, conductivity) and stability subject to the adjusted HLB value was able to be demonstrated. Because of their instability, the formulations containing PEG-8 stearate (HLB = 10.8) and polysorbate 20 were only prepared once. The ideal HLB value for surfactant in respect of the composition of an emulsion can be determined using several methods. Droplet size measurements as well as the determination of the creaming volume are the methods most commonly used [27,28]. In addition, other data such as viscosity [8], turbidity [29] or conductivity [8] can be helpful. Conductivity measurements are of interest, espe-

cially in the case of multiple W/O/W emulsions, as the transport of electrolytes between both water phases is able to be detected quite easily [11,14]. All of the formulations produced in the present work were stored for 12 weeks at RT and parameters such as droplet size, conductivity and viscosity were observed. No multiple W/O/W emulsions were created at HLB values lower than 14 and phase inversion into W/O emulsions was observed. Multiple W/O/W emulsions were created at higher HLB values. In contrast to Frenkel et al. [30], no phase inversion into O/W emulsions during the preparation process was observed at high HLB values. One parameter to greatly inuence emulsion stability is droplet size. In relation to simple emulsions, it is, in most cases, advantageous when small droplets with minimal droplet distribution are formed [8]. Reducing the size of the droplets results in better emulsion stability to gravitational separation, as described by Stokes law. The uniformity of the droplets prevents coalescence as a result of Oswald ripening. According to this information, the ideal droplet size for inner droplets in multiple W/O/W emulsions should be below 1 m. However, oil droplets must be considerably greater and in the order of magnitude of several m [17]. The inner water droplets in the primary W/O emulsions prepared in the present study were approximately 650 nm. It is assumed that, after formation of multiple droplets in the second preparation step, the inner water droplets remain unchanged. On the other hand, the oil droplet size of the multiple W/O/W emulsions is greatly dependent on the emulsiers used as well as on the HLB value of the emulsier mix. Fig. 1 shows the effect of the HLB value of a hydrophilic emulsier on the size of multiple droplets. Using steareth-10/cetosteareth-30 emulsiers, considerably smaller droplets are obtained than with PEG-8 stearate/polysorbate 20. However, despite signicant differences in droplet size, the minimum droplet size is found for both emulsier pairs at the same HLB value, i.e. approximately 1515.5. The other stability factor associated with droplet size is the change in droplet size during storage. Referring to simple emulsions, the change in droplet size is mostly associated with a coalescence process. For multiple emulsions, many more reasons could be responsible for such changes. One such reason in particular might be the ow of water between the inner and outer aqueous phases resulting from the osmotic gradient between the two sides of the oil layer. This causes the internal water drops to swell and, at the same time, increases the number of oil droplets. When the critical limit is reached, the oil layer breaks down and the inner water phase migrates into the outer water phase. As a result, the oil droplet size decreases [31].

Fig. 1. Droplet size as a function of the HLB of hydrophilic surfactant mixture for multiple emulsions containing 4% of lipophilic and 1% of hydrophilic emulsier.

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Fig. 2. Evolution of droplet sizes over time for formulations containing steareth10 and cetosteareth-30 as hydrophilic emulsiers. Droplet size is shown by way of example for one series of emulsion.

Droplet size development during storage for the formulation containing steareth-10/cetosteareth-30 is presented in Fig. 2. No change in droplet size was able to be detected. It can be assumed that, within the tested HLB range, no coalescence process occurs and that the water ow between both aqueous phases is negligible. By way of contrast, a decrease in droplet size within the whole HLB range is observed for formulations containing PEG-8 stearate/polysorbate 20. This is probably linked to the instability of ester bounds in emulsiers in the presence of electrolytes, which results in a loss of multiplicity. Conductivity measurements are commonly used for multiple emulsions to detect the release of electrolytes from the internal water phase [32,33]. The emulsions are stable when no migration of electrolytes and other substances encapsulated in the inner water phase occurs. An increase in conductivity indicates a release of electrolytes. It can occur as a result of diffusion or droplet breaking. Both processes alter the emulsion and can lead to destabilisation phenomena. The conductivity of multiple emulsions subject to the HLB value directly after preparation is presented in Fig. 3. For the emulsier mix containing steareth-10/cetosteareth-30, a minimum HLB value of approximately 1515.5 is found. For the second emulsier mix consisting of PEG-8 stearate/polysorbate 20, the minimum HLB value is observed at approximately 14.5. However, the differences in conductivity in the HLB range between 14.5 and 15.5 are very small. Irrespective of the emulsiers used, all formulations show an increase in conductivity during storage for formulations containing steareth-10/cetosteareth-30, as plotted in Fig. 4. For the emulsion

Fig. 4. Increase in conductivity over time for formulations containing steareth-10 and cetosteareth-30 as hydrophilic emulsiers. Conductivity shift is shown by way of example for one series of emulsion.

with an HLB value of 15.5, conductivity remained lowest over time compared with other formulations. This indicates that this emulsion featured the highest level of efciency in terms of electrolyte encapsulation. The creaming volume measurements are consistent with the results obtained from the measurements relating to droplet size and conductivity. The creaming of the emulsions containing cetosteareth-30 and steareth-10 as emulsiers after 3 months of storage is shown in Fig. 5. The lowest creaming volume and thus most stable formulation was found at an HLB value of approximately 15.5. The viscosity data for formulations containing steareth-10 and cetosteareth-30 are plotted as a function of the HLB (Fig. 6). The viscosity shows a maximum at HLB = 15. Recent studies of simple O/W emulsions show signicantly different characteristics [8,27]. Two areas are identied for simple O/W emulsions: one featuring a strong decrease in viscosity (low HLB values) and another featuring a linear prole, where only small changes in viscosity with increasing HLB values are observed. The HLB value, where the breaking point between both areas is estimated to be equates roughly to ideal stability. The differences between O/W and W/O/W emulsions are probably associated with the presence of an additional water phase in multiple W/O/W emulsions. Emulsion viscosity is mostly dependent on several parameters. Some of these parameters, such as, for example, the viscosity of the continuous phase, remained con-

Fig. 3. Conductivity as a function of the HLB of hydrophilic surfactant mixture for multiple emulsions containing 4% of lipophilic and 1% of hydrophilic emulsier.

Fig. 5. Creaming volume after 3 months as a function of the HLB for formulations containing steareth-10 and cetosteareth-30 as hydrophilic emulsiers.

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Fig. 6. Viscosity as a function of the HLB of hydrophilic surfactant mixture for multiple emulsions containing 4% of lipophilic and 1% of hydrophilic emulsier. Steareth-10 and cetosteareth-30 were used as hydrophilic emulsiers.

stant during the present experiment. The other parameters, such as phase ratio associated with the ow of water and electrolytes between both water phases and droplet size, were greatly inuenced by the HLB of the emulsiers used. Whereas for simple O/W emulsions the disperse-to-continuous phase ratio always remains constant, this ratio can vary for multiple W/O/W emulsions depending on the water ow between both phases. The ow from the outer water phase into the inner water phase results in an increase in viscosity as a result of a decrease in the amount of continuous phase. By way of contrast, the ow in the reverse direction leads to a decrease in viscosity. Phase inversion into an O/W emulsion is even possible above the critical weighted HLB value [30]. The electrolytes released into the outer water phase can also disturb the hydrophilic emulsier and result in a drop in viscosity. The inuence of droplet size on viscosity is described in several publications [34,35]. It is found that the reduction in droplet size results in a dramatic increase in the viscosity of concentrated W/O and O/W emulsions [35]. As shown above, the HLB of hydrophilic emulsiers can strongly inuence droplet size. Therefore, differences in viscosity because of this are expected in the HLB range studied. The viscosity therefore reects all previously described parameters such as conductivity and droplet size. It could theoretically be assumed that a maximum level of viscosity might correspond roughly to the emulsions featuring ideal parameters: droplet size and the water ow into the outer water phase are minimal and, by way of contrast, the encapsulation of electrolytes occurs on a maximum level. However, not all parameters inuence viscosity in the same way. What is more, a modied emulsion composition can also result in different viscosity characteristics. Therefore, the results obtained cannot be discussed without knowledge of other measurements. The results presented above show that methods such as droplet size measurements, conductivity and creaming behaviour could be suitable for determining the ideal HLB for hydrophilic emulsiers by means of the preparation of multiple W/O/W emulsions. Under ideal conditions, viscosity could also be helpful for this purpose. However, because viscosity projects several emulsion parameters, it can, under certain circumstances, yield ambiguous information about the stability of the formulation in question. As mentioned above, the appropriate rHLB for multiple emulsions is strongly linked to several parameters such as the HLB of the lipophilic emulsier used as well as the amount of lipophilic and hydrophilic emulsier. Therefore, the results obtained in this work are not able to be directly applied to formulations with different compositions. Generally, it must be taken into account that

the HLB of the hydrophilic emulsier in multiple W/O/W emulsions must be higher than its HLB in simple O/W emulsions. For the formulations containing parafn oil, 4% of lipophilic emulsier (HLB of approximately 4) and 1% hydrophilic emulsier, it was found that its HLB should be approximately 1515.5. In addition to determining the appropriate HLB, the clear inuence of emulsier chemistry on the stability of multiple emulsions was observed in the present study. The emulsions containing PEG-8 stearate and polysorbate 20 were characterised by very low stability. Phase separation was observed for all formulations within 4 weeks. The emulsions containing steareth-10 and cetosteareth-30 were much more stable. After 4 weeks, phase separation was found only for the formulation with an HLB value of 14. However, initial creaming was observed for all samples after 12 weeks. It can be concluded that, for the presented formulations, emulsiers containing ether groups have a positive effect on stability. By way of contrast, ester bonds have a negative effect. This might be associated with the hydrolysis of esters in the presence of electrolytes and, as a result, destabilisation of the emulsion [36]. The strong inuence of hydrophilic emulsiers on the physicochemical parameters of the emulsions is described elsewhere [6]. 4. Conclusions In the present study, the determination of the ideal HLB value for the hydrophilic emulsier in multiple W/O/W emulsions was studied. Several common techniques such as droplet size measurement, conductometric analysis, creaming volume and rheological properties were analysed and compared. It was found that most of the methods used yield similar results and are suitable for the determination of the HLB value in multiple emulsions. However, rheological data are only useful for this purpose under certain conditions. This is associated with the complexity of parameters inuencing viscosity, for example how water ow between both water phases results in changes in the disperse-to-continuous phase ratio, the amount of electrolytes released into the outer water phase or droplet size. The optimum HLB value determined for hydrophilic emulsiers for W/O/W emulsions containing 4% lipophilic (HLB = 4.3) and 1% of hydrophilic surfactant was found to range between 15 and 15.5. In addition, the strong inuence of emulsier chemistry on the stability of multiple emulsions was able to be demonstrated. In the presented formulation, hydrophilic emulsiers containing ether groups had a positive effect on stability, whereas emulsiers containing ester groups resulted in unstable emulsions. Acknowledgement We would like to thank the Hessen State Ministry of Higher Education, Research and the Arts for the nancial support within the Hessen initiative for scientic and economic excellence (LOEWEProgram) and also grant number 137/07-01. References
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